Nuclear egress of herpesviruses is an essential and conserved process in virus replication, making it an attractive target for therapeutic interventions to target the broad array of human and animal herpesviruses. The egress process is carried out by a nuclear egress complex (NEC), the core of which is composed of two conserved virus proteins called, in HSV, pUL31 and pUL34. These two proteins may recruit various other virus and host cell proteins to the NEC, the identity and function of those other proteins depending upon the species of herpesvirus. Nuclear egress requires DNA containing capsids to bud into the inner nuclear membrane, and this budding is driven by multimerization of the pUL31/pUL34 heterodimer. Several independent lines of evidence show that the pUL31/pUL34 complex is sufficient to drive this budding. Nevertheless, formation of buds without capsids is not commonly observed in TEM analysis of infected cells, suggesting that capsids must somehow trigger budding, and that capsid-less budding is inhibited in the infected cell. The mechanism of this negative regulation is not understood, and a similar knowledge gap exists for many matrix-driven virus budding processes. Herpesvirus nuclear egress can therefore provide an attractive model for understanding general principles of regulation of viral envelopment. We have previously characterized a pUL34 mutant that is deficient in nuclear egress and shows extensive formation of empty vesicles that bud into the inner nuclear membrane. This adds further evidence that the budding function of the NEC is subject to negative regulation and that this regulation can be disrupted by specific mutations in the NEC proteins. We have selected for extragenic suppressor mutants that suppress the replication defect. Interestingly, we have found two types. In one type, we see one of several mutations in the other known component of the NEC, pUL31. In the other, there are no mutations in the UL31 gene. The existence of this second class of mutants strongly suggests the participation of a third, unidentified viral gene product that is a negative regulator of budding (NRB), and provides an avenue for identification of that gene product. We will identify and characterize the function of the NRB and its interaction with the pUL31/pUL34 heterodimer by pursuing two aims: (i) Our promiscuous budding mutant contains two discrete point mutations in pUL34, and we will determine which of these is responsible for its growth and promiscuous budding phenotypes. (ii) By isolating several non-UL31 suppressors and use of whole-genome sequencing, we will identify the NRB locus. We will confirm its function using recombinant mutant viruses that carry mutant UL34 and the putative NRB suppressor mutant. We will confirm the presence of the NRB in the NEC, and then confirm that it acts as a negative regulator of budding by co-expression with pUL31 and pUL34 in the absence of other viral proteins.